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Molecular Diagnostics and Genetics |
a Author for correspondence. Fax 49-89-5160-4784; e-mail hp.schwarz{at}kk-i.med.uni-muenchen.de.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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C, Y97X). Our method also allows the
reliable, secure diagnosis of the heterozygous configuration and may
therefore be used for pre-, peri-, and postnatal diagnosis of CAH, even
when informative data of the index patient are lacking. Furthermore, it
can be used to confirm the diagnosis of CAH in newborns detected in
17-hydroxyprogesterone screening programs. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 21-hydroxylase gene (CYP21) is located in the HLA gene cluster region of chromosome 6 (6p21.3). Early genetic analyses have been performed by linkage analysis of HLA haplotypes in CAH families, with patients in the same family being HLA identical. Furthermore, associations of disease-causing alleles to certain HLA alleles have been observed (4).
Direct genetic analysis based on the characterization of the cDNA and elucidation of the genomic organization of the CYP21 genes (5)(6)(7) is complicated by the fact that both the functional gene (CYP21) and a nonfunctional pseudogene (CYP21P) are located closely adjacent in tandem arrangement with the C4A and C4B genes encoding for the fourth component of the serum complement (1)(2). The CYP21 and CYP21P genes consist of 10 exons and show a high homology with a nucleotide identity of 98% in their exon and 96% in their intron sequences.
A wide range of mutations causing 21-hydroxylase deficiency have been described thus far. Complete gene deletions, large gene conversions, single point mutations, and a small 8-bp deletion have been reported [reviewed in (2)]. The most frequent disease-causing mutations are the result of recombination events between CYP21 and CYP21P. Commonly, gene deletions are generated by unequal crossing over during meiosis, which produces a deletion of a 30-kb fragment, including the 3' end of CYP21P, the C4B gene, and the 5' end of CYP21 (8). The CYP21-inactivating point mutations are transferred in apparently small gene conversions from CYP21P to CYP21 (9). The most common point mutation is located in intron 2 and activates a cryptic splice site, producing a frameshift and early termination of a truncated protein. In a few cases de novo point mutations have been identified (2)(10)(11).
Southern blot hybridization of TaqI- and BglII-digested DNA has been described as the most efficient way for detection of gene deletions and gene conversions (2)(12). This procedure, however, must also assure that gene triplications (13)(14) and functional genes linked to the 3.2-kb TaqI band, usually associated with the pseudogene, are detected.
The highly homologous pseudogene is also a challenge in PCR-based diagnosis of point mutations in the CYP21 gene. Impure PCR products can potentially be generated by amplifying the pseudogene, with detection of mutations present in the pseudogene instead of mutations in the functional CYP21 gene. Therefore, allele-specific amplification strategies as well as pre-PCR TaqI digestion of genomic DNA have been developed to avoid the amplification of the CYP21P gene (10)(15)(16). Another diagnostic pitfall may be caused by the phenomenon of "allele dropout" as a result of unequal amplifications. This applies to a frequent point mutation in intron 2 (17).
Several methods have been described to detect mutations within the amplified PCR products: direct DNA sequencing (10)(18), allele-specific oligonucleotide (ASO) hybridization (9)(19)(20), single-strand conformational polymorphism (21)(22), ligase chain reaction (23), and amplification-created restriction sites (24)(25).
In addition to some limitations depending on the particular detection methods, the maximum overall sensitivity of mutation detection achieved by these strategies is reported to range from 90% (26) to 95% (27).
In this study we describe an improved stepwise diagnostic strategy for comprehensive mutation scanning in CAH patients. The procedure using nonradioactive Southern blotting and direct DNA sequencing was evaluated in 91 CAH patients; 181 out of 182 disease-causing alleles could be defined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fifty-eight patients suffered from the salt wasting form; 27 suffered from the simple virilizing form; and 6 suffered from the nonclassical form. The diagnosis of CAH was based on clinical classification and hormonal indicators (17-hydroxyprogesterone plasma concentration, pregnanetriol and pregnanetriolone concentrations in urine, and plasma renin activity).
southern blot analysis
Digestion of genomic DNA.
Ten micrograms of genomic DNA from
each sample was digested for 2 h with 2 U of TaqI, 2 U
of BglII, or 2 U of EcoRI/BglII
(Boehringer Mannheim), respectively, and digestion continued for at
least an additional 4 h after addition of 2 U each enzyme.
Southern blotting.
The digested DNA was separated overnight at
25 V on a 0.8% agarose gel in Loening buffer (36 mmol/L Tris-Cl, 30
mmol/L NaH2PO4, 1 mmol/L Na2EDTA)
and transferred in 0.4 mol/L NaOH for 1 h onto a positively
charged Nylon membrane (Qiagen), using the Turboblotter Rapid Downward
Transfer System (Schleicher & Schuell).
Generation of gene-specific probes.
The CYP21
probe, specific for the CYP21 and CYP21P genes,
was PCR-amplified from genomic DNA, using primers 21OH-probe-F and
CAH-1R (Table 1
). The C4 probe, specific for the C4A and
C4B genes, was amplified from cDNA with primers C4-F and
C4-R (Table 1
). The utilized cDNA was generated by reverse
transcription of total RNA using the First-Strand cDNA synthesis kit
(Pharmacia Biotech) and the primer C4-R. Total RNA was prepared from
liver tissue using the RNeasy total RNA kit (Qiagen) according to the
manufacturer's instructions.
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The PCR products were subcloned in the pGEM-T vector (Promega), and the clones were checked by sequencing.
Plasmid DNA (200 ng) was labeled with digoxigenin (DIG), using the PCR DIG probe synthesis kit (Boehringer Mannheim) according to the manufacturer's protocol, with 1.5 mmol/L MgCl2, 2 U of Taq DNA polymerase, and 10 pmol of each amplification primer (see above). PCR was performed as follows: initial denaturation at 94 °C for 7 min, 30 cycles at 94 °C for 45 s, 53 °C for 1 min, 72 °C for 2 min, and final extension at 72 °C for 7 min. The labeled probes were purified with the QIAquick PCR purification kit (Qiagen).
Hybridization and immunological detection.
The
TaqI-digested DNA was hybridized simultaneously with the
CYP21 and the C4 probes. BglII- and
EcoRI/BglII-digested genomic DNAs were hybridized
with the CYP21 probe only. The hybridization and
immunological detection of the DIG-labeled nucleotides were performed
using the DIG luminescent detection kit and a protocol according to
Engler-Blum et al. (28) with the following modifications:
The purified probes were used in a concentration of 5 ng DIG-labeled
probe/mL hybridization solution; anti-DIG alkaline phosphatase Fab
fragments (Boehringer Mannheim) were diluted 1:20 000 in blocking
buffer (0.1 mol/L maleic acid, 3 mol/L NaCl, 30 mL/L Tween 20, 5 g/L
blocking reagent); CDP-starTM chemiluminescent substrate
(Boehringer Mannheim) was diluted 1:100 with substrate buffer (0.1
mol/L Tris-Cl, 0.1 mol/L NaCl, 50 mmol/L MgCl2). The
membranes were exposed to x-ray films for 15–20 min.
pcr amplification and dna sequencing
A quantitative TaqI restriction digestion was performed
before the PCR to amplify exclusively the functional CYP21
gene associated with the 3.7-kb TaqI fragment. A
quantitative BclI digestion was needed for amplification of
the functional CYP21 gene linked to a 3.2-kb TaqI
band. Genomic DNA was digested under the same conditions as described
for Southern blotting.
The Expand long-template PCR system (Boehringer Mannheim) was used for PCR amplification. Reactions were carried out in a volume of 50 µL containing 5 µL of 10x PCR reaction buffer, 2.5 U of DNA polymerase mix, 400 µmol/L each dNTP, 10 pmol of primers CAH-F and CAH-R, and 200 ng of TaqI- or BclI-digested genomic DNA, respectively. Cycling conditions for PCR were as follows: initial 5 min at 94 °C, 30 cycles at 94 °C for 30 s, 53 °C for 30 s, 68 °C for 3 min, and final extension at 68 °C for 7 min.
After amplification, 10 µL of PCR product was digested with 1 U of BclI or TaqI restriction enzyme, respectively, and separated on a 0.8% agarose gel in Tris-borate-EDTA buffer (84 mmol/L Tris-Cl, 90 mmol/L boric acid, 2 mmol/L Na2EDTA) for 1 h.
Two allele-specific fragments, AS1 and AS2, were amplified in a
heminested primer PCR using 1 µL of 1:100 diluted PCR product
(generated from BclI-digested genomic DNA) as template. The
amplifications were performed with the Expand high fidelity PCR system
(Boehringer Mannheim) in a reaction volume of 50 µL containing 5 µL
of 10x PCR buffer 1, 1 mmol/L MgCl2, 200 µmol/L each
dNTP, 2.5 U of DNA polymerase mix, and 10 pmol of each primer. AS1 was
generated using primers CAH-F and AS1-R. AS2 was produced using primers
AS2-F and CAH-R (Table 1
). Cycling conditions were the same as
described above.
The PCR products were purified with the QIAquick PCR purification kit
(Qiagen) and sequenced using the ABI dye terminator cycle sequencing
technique with nested primers (Table 1
). The reaction volume was 10
µL containing 4 µL of Ready Reaction Dye Terminator Kit
(Perkin-Elmer), 10 pmol of each primer, and 3 µL of purified PCR
product. Cycle DNA sequencing was performed as follows: 25 cycles of
94 °C for 15 s, 55 °C for 15 s, and 60 °C for 4 min.
The samples were separated on an Applied Biosystems PRISM 377 DNA
Sequencer and analyzed using the ABI PRISM Sequence Navigator.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, lane 1). The cohybridized probe specific for the C4
gene recognizes a 7.0-kb fragment arising from the C4A gene
and either a 6.0- or a 5.4-kb band linked to the C4B
gene because of an intron polymorphism in the C4B gene. In
lane 1 of Fig. 1
, a heterozygous state for this polymorphism is shown.
A deletion of the CYP21 gene in a heterozygous state leads
to a decrease in intensity of the 3.7-kb band (Fig. 1
, lane 4), which
is completely absent in a homozygous state (Fig. 1
, lane 7). In
addition, the 12-kb BglII bands, which bear the
CYP21 pseudogene and the fragments specific for the
C4B gene, are also less intense or completely missing (Fig. 1
, lanes 2, 5, and 8). This type of gene alteration was disclosed in a
total of 43 disease alleles (Table 2
). In contrast, gene conversions do not reduce the intensity of
the BglII band. Eleven alleles of this gene conversion
mutation type could be identified.
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The EcoRI/BglII double restriction digestion also
allowed the differentiation between a 2.5-kb functional and a 2.0-kb
nonfunctional fragment (Fig. 1
, lanes 3, 6, and 12). Usually the dosage
ratio between 3.7-kb and 3.2-kb TaqI fragments is roughly
equal to the ratio between 2.5-kb and 2.0-kb
EcoRI/BglII fragments. A discrepancy between
these two ratios indicates a functional gene associated with the 3.2-kb
TaqI fragment. In three unrelated simple virilizing CAH
patients, complete loss of the 3.7-kb TaqI fragment and
clearly visible 2.5-kb EcoRI/BglII fragments were
identified. This obviously indicated a functional 21-hydroxylase gene
associated with a 3.2-kb TaqI fragment, which usually is
associated with the pseudogene.
The possible simultaneous deletion of a pseudogene in conjunction with a deletion of the functional gene probably could prevent a correct interpretation of the results. This situation, however, was unraveled by hybridization with the C4-specific probe, yielding the appearance of a novel 6.4-kb fragment and a less intense 7.0-kb band.
By Southern blot analysis, gene deletions were detected in 23.6% of
our cohort and gene conversions were detected in 6%. The subsequent
systematic procedure for the detection of point mutations delineated
from the results of the Southern blot analysis is schematically
depicted in Fig. 2
.
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detection of point mutations
Depending on the results of the Southern blot analysis, the
detection of point mutations was performed in two different ways (Fig. 2A
).
If a functional gene indicated by a 3.7-kb TaqI fragment was
detected in the Southern blot analysis, a 4-step procedure was
subsequently performed (Fig. 2B
1). The CYP21 gene was
amplified after TaqI restriction digestion of the genomic
DNA, which yielded a 3.5-kb PCR product. To control for coamplification
of the pseudogene, this product was then digested with BclI.
A single base pair substitution changes a TaqI to a
BclI restriction site and vice versa. If only
CYP21 was amplified, BclI digestion of
CYP21 produced two fragments (0.5 and 3.0 kb). An undigested
3.5-kb fragment appeared if CYP21P was coamplified. After
the control digestion, CYP21 PCR products were sequenced in
six overlapping sequencing reactions covering all exons and introns. In
cases of a heterozygous 8-bp deletion in exon 3, the primer CAH-2E-F
had to be used additionally, because the resulting frameshift made
sequence analysis upstream the deletion impossible.
Another procedure had to be performed (Fig. 2B
2) in the rare case that
a functional gene associated with a 3.2-kb TaqI fragment was
detected in the Southern blot analysis (Fig. 1
, lanes 10, 11, and 12).
Genomic DNA was digested with BclI (step 1) before
amplification with CAH-F and CAH-R to eliminate the CYP21
gene associated with the 3.7-kb TaqI fragment (step 2). The
resulting 3.5-kb PCR product consisted of a mixture of
CYP21P and CYP21 associated with a 3.2-kb
TaqI fragment. A TaqI control digestion (step 3),
yielding two fragments (0.5 kb and 3.0 kb) assured the completeness of
the BclI digestion before amplification. In the absence of a
3.7-kb TaqI fragment in the Southern blot analysis, these
three steps were not necessary.
Two allele-specific primers (AS1-R and AS2-F) were designed for the amplification of functional genes associated with a 3.2-kb TaqI band. These primers were specific for the wild-type allele, which did not include the 8-bp deletion in exon 3. The AS1 fragment (1.4 kb) was amplified with CAH-F and AS1-R; the AS2 fragment (2.1 kb) was amplified with AS2-F and CAH-R. AS1 was sequenced with CAH-2E-F and CAH-2R, AS2 with CAH-4R, CAH-6R, CAH-7R, CAH-9R, and CAH-R.
If both a 3.2-kb TaqI band and a 3.7-kb TaqI
fragment were detected, the procedures depicted in Fig. 2B
1 and 2B2 had
to be performed.
By utilizing this combined strategy of Southern blotting analysis and direct DNA sequencing of the 21-hydroxylase gene in our CAH disease cohort, we were able to unravel the molecular lesions in 181 out of 182 alleles (diagnostic sensitivity >99%).
All known point mutations arising by the mechanism of apparent gene
conversion were detected (Table 3
). Furthermore, a new mutation in exon 3 (bp 670, CA, Y97X)
was revealed compound heterozygous with the intron 2 splice site
mutation in a patient who suffered from the salt wasting form (Fig. 3
). The only case in which we could not identify an inactivating
point mutation was a patient with salt wasting CAH carrying a
heterozygous deletion allele. In three unrelated simple virilizing
patients, the P30L mutation was found responsible for the partial
inactivation of the functional gene associated with a 3.2-kb
TaqI fragment.
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Approximately 84% of disease-causing alleles were identified by Southern blotting together with only a single direct DNA sequencing reaction because the CAH-4R sequencing ladder covered the frequent point mutations (A/C to G in intron 2, 8-bp deletion in exon 3, and the I172 N mutation).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The main initial tool for genotyping CAH patients is the Southern blot
analysis. Southern blotting after TaqI and BglII
digestion of genomic DNA has been described earlier as sufficient for
detection of gene deletions and gene conversions
(2)(12) (Table 2
). By using the
EcoRI/BglII restriction digestion, we added a
feature that enabled us to distinguish functional from nonfunctional
genes. The CYP21-specific probe was designed to give
full-length hybridization with all the CYP21 and
CYP21P associated bands. This avoids potential pitfalls in
interpretation arising from only partial hybridization that would give
erroneous band ratio. Adapting the nonradioactive Southern blot
(28) to CYP21 gene analysis proved to be
advantageous compared with the radioactive procedure. By using the
chemiluminescence substrate CDP-star, exposure times of the blots are
shortened to 15–20 min instead of the 3- to 4-day exposure necessary
in using radioactive Southern blot analysis. The chemiluminescence
procedure also allows repeated exposition of the x-ray films until an
optimal result for analysis is achieved.
In our opinion, Southern blot analysis is necessary to distinguish compound heterozygosity whenever one allele carries a deletion or conversion of the CYP21 gene. Without Southern blot analysis, such cases would appear as homozygotes for the mutation detected in DNA sequencing (point mutations, 8-bp deletion).
The maximum sensitivity in CAH mutation detection reported thus far in other studies ranges from 90% (26) to 95% (27). Methods for detection of point mutations, such as ASO hybridization (9)(19)(20), amplification-created restriction sites (24)(25), or ligase chain reaction (23), can only find mutations already known. By single-strand conformational polymorphism analysis (21)(22), it is difficult to detect all de novo mutations because the optimal conditions for the detection of various mutations usually vary and need to be established separately. In this study >99% of the defective alleles were elucidated. Furthermore, methods such as ASO hybridization, amplification-created restriction sites, and single-strand conformational polymorphism are difficult to perform in parallel. In allele-specific amplifications, many PCR reactions using different primers and annealing temperatures must be performed on different thermal cyclers or at different times, respectively. In ASO techniques, different hybridization temperatures are needed for detecting different mutations. In our experience, the direct DNA sequencing method for CAH point mutation detection is easier to handle because the six DNA sequencing reactions vary only in the use of primers, whereas all other reaction conditions are unchanged. In addition, known as well as new mutations are recognized.
Another major issue in the mutation analysis of the CYP21 gene is the phenomenon of allele dropout (17). We also observed this phenomenon using nested primer PCR for amplification of a fragment including exon 3 and its intron boundaries. Although there was no base change in the forward and reverse primer binding sites, the C alleles at bp 656 were not amplified (not shown). This is the major reason that procedures using multiple nested primer PCR (16) can lead to false interpretation of the analytical result.
The analytical strategy for CAH mutation scanning applied in this study led to a successful elucidation of the molecular cause of the disease in 181 out of 182 unrelated alleles. A total of 91 clinically and biochemically characterized patients were characterized. We were able to identify all classical known, disease-causing mutations of the 21-hydroxylase gene in a frequency similar to other studies (2)(27)(29)(30) Furthermore, it was possible to detect a novel mutation located in the genomic sequence (Y97X).
One female patient, suffering from the salt wasting form, was found to be only heterozygous for a gene deletion. No additional known or novel inactivating mutation in the encoding region of the CYP21 gene located on the other allele was found. No mutation was detected inside the intron sequences, which might cause an activated cryptic splice site.
Highly sensitive and reliable mutation scanning of the CYP21 gene also supports the diagnosis of the heterozygous configuration and may therefore represent the gold standard in prenatal diagnosis of CAH. Because prenatal dexamethasone therapy of the affected fetus is effective (31)(32), exact genotyping of the unborn is important. Our method can be used for pre-, peri-, and postnatal diagnosis of CAH caused by 21-hydroxylase deficiency, even in situations when informative data of the index patient are lacking. Furthermore, it can be used to confirm the diagnosis of CAH in newborns detected in 17-hydroxyprogesterone screening programs.
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Acknowledgments |
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Footnotes |
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1 Nonstandard abbreviations: CAH, congenital adrenal
hyperplasia; ASO, allele-specific oligonucleotide; and DIG,
digoxigenin. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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